The central serotonergic system is the target of several successful psychotherapeutic drugs, but progress in its study has frequently been impeded because of a lack of selective drugs. The objective of the proposed research is to develop a technique to selectively attenuate the expression of the serotonin 5-HT2A receptor using an antisense oligonucleotide complementary to specific mRNA's, in cultured cells an in vivo. Antisense oligonucleotides act as specific inhibitors of gene expression by inhibiting transcription and/or the translation of mRNA. Experiment 1 will examine the specificity of antisense treatment in affecting the expression of 5-HT2A receptors in C6 cells, the dose response for inhibition of expression, and the time for resynthesis. Oligonucleotides in the sense, antisense and nonsense configurations will be tested for their ability to selectively attenuate the expression of 5- HT2A receptors in cultured C6 glioma cells. Cells will be incubated in the presence of the oligonucleotides and receptor gene transcription will be measured using an RNase protection assay and radioligand binding will be used to estimate receptor expression. Experiment 2 will examine the in vivo specificity of antisense treatment on 5-HT2A receptors in rat brain. Rats will be infused intraventricularly (using Alzet Minipumps) with the three configurations of oligonucleotides. The dose- and time-response for receptor loss and resynthesis will then be determined in rat brain. 5-HT2A receptor binding will be determined autoradiographically and mRNA levels for the 5-HT2A receptors will be determined by in situ hybridization. To verify the specificity of the antisense treatment, the closely related 5- HT2C receptor will also be measured., Experiment 3 will characterize the physiological expression of 5-HT2C receptor will also be measured. Experiment 3 will characterize the physiological expression of 5-HT2A receptor downregulation. The effects of prior antisense treatment ion the corticosterone response to immobilization stress will be assessed. Animals will be immobilized for 60 min and tail blood obtained for the determination of plasma corticosterone (assessed using radioimmunoassay). To better define the respective roles of the 5-HT2A and 5-HT2C receptors, the effect of 5-HT2A receptor attenuation on behavior int he elevated plus- maze and on behavioral responses to the 5-HT2A receptor attenuation on behavior int he elevated plus-maze and on behavioral responses to the 5- HT2A agonist DOI will be determined. The proposed work will help define the physiological role of the 5-HT2A receptor and provide a basis for the development of more efficacious pharmacotherapies for posttraumatic stress disorder, anxiety, and depression.
